中國農業科學 ?? 2019, Vol. 52 ?? Issue (7): 1237-1246.doi: 10.3864/j.issn.0578-1752.2019.07.011

? 植物保護 ? 上一篇    下一篇

褐飛虱GSK-3調控糖原與海藻糖代謝的潛在功能

丁艷娟1,劉永康1,羅雨嘉1,鄧穎梅1,徐紅星2,唐斌1(),徐彩娣1,3   

  1. 1 杭州師范大學生命與環境科學學院,杭州 310036
    2 浙江省農業科學院植物保護與微生物研究所,杭州 310021
    3 杭州師范大學教育學院,杭州 310036
  • 收稿日期:2018-11-15 接受日期:2018-12-29 出版日期:2019-04-01 發布日期:2019-04-04
  • 通訊作者: 唐斌 E-mail:[email protected]
  • 作者簡介:丁艷娟,E-mail: [email protected]
  • 基金資助:
    國家自然科學基金(31672081);國家自然科學基金(31371996);國家級大學生創新創業訓練計劃項目(201810346031)

Potential Functions of Nilaparvata lugens GSK-3 in Regulating Glycogen and Trehalose Metabolism

DING YanJuan1,LIU YongKang1,LUO YuJia1,DENG YingMei1,XU HongXing2,TANG Bin1(),XU CaiDi1,3   

  1. 1 College of Life and Environmental Science, Hangzhou Normal University, Hangzhou 310036
    2 Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021
    3 College of Education, Hangzhou Normal University, Hangzhou 310036
  • Received:2018-11-15 Accepted:2018-12-29 Online:2019-04-01 Published:2019-04-04
  • Contact: Bin TANG E-mail:[email protected]

摘要:

【目的】昆蟲胰島素信號途徑能夠介導糖原合成酶激酶3(glycogen synthase kinase 3,簡稱GSK-3或GSK3)調控體內糖原及海藻糖等糖代謝過程,從而控制昆蟲的各項生命活動。論文旨在探究糖原合成酶激酶在褐飛虱(Nilaparvata lugens)體內對糖原與海藻糖代謝的調控作用。【方法】首先,基于GSK-3的cDNA編碼序列,利用ExPASy工具翻譯GSK-3氨基酸序列,預測蛋白分子量大小及等電點(pI);然后利用SignaIP4.1Server對其信號肽進行分析。其次,以筆者實驗室飼養的褐飛虱為研究對象,從4齡開始,每12 h取材,取至成蟲48 h。利用Trizol法提取褐飛虱總RNA,根據反轉錄試劑盒合成第一鏈DNA,以18S作為內參基因,通過實時熒光定量PCR(qRT-PCR)檢測褐飛虱GSK-3在不同齡期mRNA水平上的相對表達量。然后利用RNAi技術,向褐飛虱體內顯微注射雙鏈RNA(dsRNA)抑制GSK-3,以注射dsGFP的褐飛虱作為對照組。注射后48 h利用qRT-PCR技術檢測GSK-3的表達情況,確定抑制效果。另外,取注射后48 h蟲體,分別測定褐飛虱體內海藻糖、葡萄糖、糖原含量及海藻糖酶(trehalase,TRE)活性變化。最后采用qRT-PCR檢測胰島素信號通路胰島素受體基因(insulin receptor,InR)、類胰島素多肽基因(insulin-like peptides,Ilps)及海藻糖代謝途徑TRE、海藻糖合成酶基因(trehalose-6-phophate synthase,TPS)、糖原磷酸化酶基因(glycogen phosphorylase,GP)、糖原合成酶基因(glycogen synthase,GS)中相關基因的表達,分析GSK-3在胰島素信號通路及海藻糖代謝途徑中的調控作用。【結果】褐飛虱GSK-3開放閱讀框為1 914 bp,編碼637個氨基酸;預測蛋白分子量為69.25 kD,等電點為9.15,為偏堿性蛋白,無信號肽結構,序列高度保守。發育表達模式結果顯示GSK-3在不同發育階段表達不一致,5齡若蟲蛻皮前后低表達。GSK-3的dsRNA注射后48 h,與對照組dsGFP相比,GSK-3表達極顯著下降,表明RNA干擾效果明顯。糖原含量和兩類海藻糖酶活性顯著下降,而海藻糖含量顯著上升,推測糖原和葡萄糖轉化為海藻糖,作為其生理活動的能量來源。qRT-PCR檢測發現,當GSK-3表達抑制后48 h,TRE1-2的表達量顯著下降,而TRE1-1TRE2的表達量極顯著下降。另外,2個TPS基因、GS以及GP的表達量均極顯著下降;胰島素信號通路的2個InR基因和4個Ilps基因的表達同樣被抑制,間接表明InR能夠調控GSK-3的表達。【結論】褐飛虱GSK-3低表達后能夠通過調控胰島素信號通路及海藻糖代謝途徑相關基因表達來調控糖原及海藻糖代謝。相關研究結果有助于更加全面地探索褐飛虱等昆蟲糖原合成酶激酶調控海藻糖及糖類物質平衡的潛在分子機理。

關鍵詞: 褐飛虱, RNA干擾, 糖原合成酶激酶3, 糖原與海藻糖代謝, 實時熒光定量PCR(qRT-PCR)

Abstract:

【Objective】The insect insulin signaling pathway can mediate glycogen synthase kinase 3 (GSK-3 or GSK3) to regulate glucose metabolism in the body, such as glycogen and trehalose, thereby controlling different life activities of insects. The objective of this study is to explore the regulation of glycogen synthase kinase on the metabolism of glycogen and trehalose in Nilaparvata lugens.【Method】 Firstly, based on the cDNA coding sequence of GSK-3, the GSK-3 amino acid sequence was translated using the ExPASy tool to predict the molecular weight and isoelectric point (pI) of the protein, and then the signal peptide was analyzed by SignalaIP4.1Server. Secondly, the N. lugens raised in the author’s laboratory was collected every 12 hours from the 4th instar to 48-h-old adult. The total RNA of N. lugens was extracted by Trizol method. The first strand DNA was synthesized according to the reverse transcription kit, and 18S was used as the internal reference gene. The relative expression of GSK-3 in different ages at mRNA level was detected by quantitative real-time PCR (qRT-PCR). Then, double-stranded RNA (dsRNA) was microinjected into N. lugens with RNAi technology to inhibit the GSK-3, and N. lugens of dsGFP was injected as a control group. The expression of GSK-3 was detected by qRT-PCR 48 h after injection to determine the inhibitory effect. In addition, the N. lugens was taken 48 h after injection, and the change of trehalose, glucose, glycogen content and trehalase (TRE) activity in N. lugens was determined. Finally, the relative expression of related genes in insulin signaling pathway (including insulin receptor (InR), insulin-like peptides (Ilps)) and trehalose metabolism pathway (TRE, trehalose-6-phophate synthase (TPS), glycogen phosphorylase (GP), glycogen synthase (GS)) was detected by qRT-PCR to analyze the regulation of GSK-3 in insulin signaling pathway and trehalose metabolic pathway.【Result】The open reading frame of N. lugens GSK-3 is 1 914 bp, encoding 637 amino acids; the predicted molecular weight of the protein is 69.25 kD, and the isoelectric point is 9.15. It is a basic protein with no signal peptide structure and the sequence is highly conserved. The results of developmental expression pattern showed that the expression of GSK-3 was inconsistent at different developmental stages, and the expression was low before and after molting of 5th instar nymph. At 48 h after the dsRNA injection of GSK-3, the expression of GSK-3 decreased significantly compared with the dsGFP of the control group, indicating that the RNA interference effect was obvious. Glycogen content and two types of trehalase activity decreased significantly, while trehalose content increased significantly. It is speculated that glycogen and glucose are converted to trehalose as an energy source for their physiological activities. The results of qRT-PCR showed that the expression of TRE1-2 significantly decreased 48 h after the inhibition of GSK-3 expression, while the expression of TRE1-1 and TRE2 extremely significant decreased. In addition, the expression of two TPS genes, GS and GP genes all extremely significant decreased; the expression of two InR genes and four Ilps genes in the insulin signaling pathway were also inhibited, indirectly indicating that InR can regulate the expression of GSK-3. 【Conclusion】 The low expression of GSK-3 in N. lugens can regulate glycogen and trehalose metabolism by regulating insulin signaling pathway and trehalose metabolism pathway related gene expression. The relevant research results will help to explore more comprehensive molecular mechanisms for the regulation of the balance of trehalose and carbohydrates by insect glycogen synthase kinases such as N. lugens.

Key words: Nilaparvata lugens, RNA interference (RNAi), glycogen synthase kinase 3, glycogen and trehalose metabolism, quantitative real-time PCR (qRT-PCR)

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